The use of the effective fraction of alkaloids from mulberry twig in preparing hypoglycemic agents

ABSTRACT

The present invention relates to an effective fraction of alkaloids and the effective fraction is prepared from mulberry twig and its active ingredients are a composition of alkaloids. Determined by HPLC, the percentage of the total alkaloids are 50% or more by weight in the effective fraction and the percentage of the compound 1-deoxynojirimycin is 30% or more by weight in the total alkaloids. The effective fraction of the invention is prepared as the following steps: the mulberry twig is extracted by solvents, and the extract is precipitated by alcohol precipitation or flocculation to remove the impurities, and then concentrated, purified by resin chromatography. The present invention also relates to a pharmaceutical composition containing the said effective fraction of alkaloids and to the use of the effective fraction of alkaloids according to claim  1 - 4  in preparing hypoglycemic agents.

FIELD OF THE INVENTION

The present invention relates to use of the effective fraction ofalkaloids from mulberry twig for preparation of hypoglycemic agents.

DESCRIPTION OF RELATED ART

It is reported that the incidence of diabetes mellitus is up to 4% inthe people over age of 40 and more than 11% in the people over age of 60in China. According to the latest data from the WHO, the number ofdiabetic patients will quadruple in the next 15 years in China. Diabeteshas become a serious disease that threatens human life and health. Morethan 90% of the patients with diabetes mellitus are suffered from type 2diabetes and these patients have to take long-time oral hypoglycemicagents to control the hyperglycemia. At present, there are five classesof commercial oral hypoglycemic drugs including: (1) sulfonylureas, (2)biguanides, (3) α-glucosidase inhibitors, (4) insulin-sensitizing agent,(5) traditional Chinese medicine. In China, the annual sales of oralhypoglycemic drugs are more than 10 billion yuan.

In 1950s, oral hypoglycemic drugs such as sulfonylureas and biguanidecame into the market, which enabled the patients with type 2 diabetes tocontrol the blood glucose levels, prolong longevity and improve thequality of life. However, sulfonylurea oral agents tend to inducehypoglycemia and biguanides agents may cause serious side effects ongastrointestinal tract. After 40 years of effort, a new type ofα-glucosidase inhibitor was found for the treatment of type 2 diabetesmellitus. The application of such agent has been recognized as a new wayto control diabetes all over the world. The α-glucosidase inhibitorinteracts with α-glucosidase located in the brush border of the smallintestine. It can slow down the hydrolization of polysaccharides intomonosaccharides, delay the absorption of glucose, postpone thepostprandial blood sugar peaks, and reduce the range of “blood sugarfluctuations”. Therefore, α-glucosidase inhibitor can reduce bloodsugar, prevent and delay the development of certain chroniccomplications of diabetes. At the beginning of 1990s, the German BayerCompany introduced the new drug glucobay (Acarbose), which rapidly cameinto the markets of more than 50 countries and obtained a goodtherapeutic effect. In 1994, glucobay entered the Chinese market andafter ten years of clinical practice, it has demonstrated to beeffective and safe in the treatment of type 2 diabetes mellitus.Moreover, it has received a good social benefits and significanteconomic benefits.

There are abundant medicinal plants in China and there are numeroushypoglycemic crude preparations, while whose mechanisms of action areunknown and whose qualities are not controllable. Mulberry twig is thedry branch of morus alba, which contains hundreds of compounds,including polysaccharide, monosaccharide, vegetable protein, phenols,falconoid, saponin, organic acid, amino acid, polyhydroxy alkaloids,etc. In 1990s, Institute of Materia Medica, Chinese Academy of MedicalSciences discovered that the water and/or ethanol extract of mulberrytwig had a strong α-glucosidase inhibitory activity and then applied fora Chinese patent “the application of ethanol and/or aqueous extract ofMulberry twig in preparation of hypoglycemic agent” (application NO.97112359.4), which has been granted Apr. 10, 2002 by State IntellectualProperty Office of PRC. As the dosage of crude preparation from ethanoland/or aqueous extract is too high and the quality is not controllable,Institute of Materia Medica, Chinese Academy of Medical Sciences carriedout further studies on effective fraction of mulberry twig.

In addition to the aforementioned patent application 97112359.4, weretrieved another 3 pieces of patents related to mulberry extract withα-glucosidase inhibitor activity including:

A Chinese patent application 01113191.8, “Chinese medicinal extract withα-glucosidase inhibitor activity, and its preparation and application”,disclosed a Chinese medicinal extract which is the total alkaloid fromMorus alba L., mulberry leaves and mulberry fruit. It didn't refer tomulberry twig and had no description of the component of the alkaloid.

A Chinese patent application 02113004.3, “Extract of mulberry twig andits preparation and its novel use”, referred to the total flavones butnot to alkaloids. The extract was used to prevent hyperuricemia and goutbut not diabetes.

A Chinese patent application 200410018677.4, “Medicinal compositionpossessing α-glucosidase inhibitor activity and its use”, disclosedmedicinal composition consisted of alkaloids and flavones. Thealkaloids, whose weight ratio is 5-95%, are total alkaloids extractedfrom silkworm excrement, mulberry twig, Morus alba L. or mulberry leavesand the flavones extract, whose weight ratio is 10-95%, is at least onecompound selected from the group consisting of catechin, quercetin andtea polyphenols. It didn't specifically disclose the content of totalalkaloids in the alkaloid extract. In addition, the use of thecombination of alkaloid extract and flavone extract in therapy onlyshowed dose-additive effect, rather than have synergism under theexperiment conditions.

Although Morus alba L. has highest content of alkaloids, but its plantresource is rare and the collection will bring damage to the plant.Mulberry leaf, which contains the lowest content of alkaloid, is hard toextract because it contains many impurities. There are abundantresources of Mulberry twig, which has moderate content of alkaloidbetween Morus alba L. and mulberry leaf, and it can be easily got byannually pruning. In addition, its price is inexpensive and its qualityis controllable. Therefore, it has great practical significance to studythe hypoglycemic effect of the effective fraction of alkaloids frommulberry twig.

DETAILED DESCRIPTION OF THE INVENTION

In view of the problems in crude preparation from ethanol and/or aqueousextract, the present invention takes a further research on the effectivefraction of alkaloids from mulberry twig. Through the studies ofα-glucosidase inhibitor activities of different extract fractions frommulberry twig, we discover that the water-soluble alkaloids show thestrongest activity. In other words, the effective fraction withhypoglycemic activity exists in water-soluble alkaloids.

This invention is a further technical improvement based on CN97112359.4.

In order to achieve the purpose of the present invention, the inventionuses the following technical solutions:

An effective fraction of alkaloids of the present invention is preparedfrom mulberry twig and the active ingredients are a composition ofalkaloids; the content of the total alkaloids are 50% or more by weightin the effective fraction and the content of the compound1-deoxynojirimycin is 30% or more by weight in the total alkaloids.

A preferred effective fraction of the invention includes at least onecompound selected from the group consisting ofN-methyl-1-deoxynojirimycin, fagomine, 3-epi-fagomine,1,4-dideoxy-1,4-imino-D-arabinitol, 1,4-dideoxy-1,4-imino-D-ribitol,calysteginB₂, 2-O-(α-D-galactopyranosyl) -1-deoxynojirimycin,6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

In a more preferred effective fraction of the invention, the percentageof the compound 1-deoxynojirimycin is 40% or more by weight in the totalalkaloids.

In the most preferred effective fraction, the percentage of the compound1-deoxynojirimycin is 50% or more by weight in the total alkaloids.

The present invention also discloses a method for preparing theeffective fraction of alkaloids, that is: taking mulberry twigs asmedicinal material, after smashing, extracting, separating, purifyingand concentrating, the effective fraction, in which the content of thetotal alkaloids are 50% or more by weight, is prepared.

A method for preparing the effective fraction of alkaloids is describedin detail as follows:

(a) the mulberry twig was extracted by solvents and the extract solutionwas concentrated, precipitated and removed impurities;

(b) the supernatant fluid was added into cation exchange resin andeluted with weak base eluent; and

(c) the eluent was concentrated and added into anion exchange resin andthe part not absorbed was collected, and dried to get the effectivefraction powder.

Step (a) conducts as follows:

The said mulberry twig is preferably to be fresh one;

The preferred mulberry twig is dried and properly smashed in order toincrease the contact areas with solvents and improve the efficiency.

The solvents are selected from the group consisting of water, alcohol,the mixture of water and alcohol, and acidic water; the preferredalcohols include methanol, ethanol, iso-propanol, butanol, etc.; themost preferred solvent is water.

The amount of the solvent is 4-12 times of medicinal material and theextraction is repeated 1-3 times. The extraction can be carried outunder static or dynamic conditions, preferably under dynamic conditions.In order to improve the efficiency of extraction, ultrasound can beused. The extraction is conducted at temperatures range from roomtemperature (eg 20° C.) to the solvent's reflux temperature, preferablyat reflux temperature. The extraction process can be continuous orintermittent, wherein the intermittent process can be repeated 1-3times. The extraction time ranges from 1 hour to 4 hours, preferablyfrom 1.5 hours to 2.5 hours.

The precipitation prefers to be alcohol precipitation or flocculation,and 50-80% alcohol is used for flocculation.

Removal of impurities prefers by way of filtering or centrifugation.

Step (b) conducts as follows:

After adding the supernatant fluid into cation exchange resin, the resinis selectively washed with distilled water to remove the impurities notabsorbed, before elunting with weak base water. This can improve thepurity.

The preferred eluent is ammonia solution, whose concentration ispreferably to be 0.2-1N.

Cation exchange resins are selected from sulfonic strong acid type andcarboxylic week acid type; sulfonic strong acid type is preferred; themost preferred is 001×7(732#) cation resin.

The weight ratio of the crude drug and cation exchange resin is1:0.1-0.5; more preferred to be 1:0.1-0.4; most preferred to be1:0.2-0.3.

Step (c) conducts as follows:

After adding the concentrated eluent into anion exchange resin, theresin can be selectively washed by distilled water to collect all thecompounds not absorbed and improve the final yield.

The drying methods include vacuum concentration, spray drying and freezedrying.

Anion exchange resins could be strong base type, such as quaternaryamine (also known as quaternary amine) —NR₃OH(R is hydrocarbon group) orweek base type, such as primary amine-NH₂, secondary amine-NHR, andtertiary amine —NR₂; preferably to be quaternary amine strong base type.

The most preferred resin is Dowex 1×2(OH⁻) Anion exchange resin or201×7(717, OH⁻) Anion exchange resin.

The weight ratio of the crude drug and anion exchange resin is1:0.2-0.8; preferred to be 1:0.3-0.7; most preferred to be 1:0.5-0.6.

Materials for ion exchange resin include styrene series, acrylic acidseries, phenolic(FP) series, epoxy(EPA) series, vinyl pyridine(VP)series and urea-formaldehyde(UA) series; preferably to be styreneseries.

The styrene resins mentioned here include gel type and macroporous type;the gel type is preferred.

The term “crude drug” mentioned in the present invention means thestarting material mulberry twig.

A preferred preparation method is as follows:

(a) to every 350 kg of fresh mulberry twig, 2000 L of water is added andthe mixture is refluxed for 2 hours, the extraction process was repeated2 times, the extractions were combined and concentrated to a volume of250 L. 250-270 L of ethanol is added to the concentrate. The mixture isprecipitated for 24 hours and filtered;

(b) the supernatant fluid is added into 001×7(732#) cation exchangeresin, whose dosage is crude drug: resin=1:0.2-0.3 by weight, washedwith distilled water to neutral, then washed with 0.5N ammonia solution,and the eluent is collected;

(c) the eluent is added into Dowex 1×2(OH⁻) Anion exchange resin or201×7(717, OH⁻) Anion exchange resin, whose dosage is crude drug:resin=1:0.5-0.6 by weight and the part not absorbed is collected andconcentrated via vacuum concentration and dried to get a light brownpowder.

Quantitative Determination of the Effective Fraction

As the alkaloids of the effective fraction are consisting of polyhydroxyalkaloids and their glycosides, which have no UV absorption, hence theyhave to be derived by derivatization reagents and then separated byHPLC. It is proved that this determination method have strongspecificity, high sensitivity, and excellent repeatability.

It is found that in one class of alkaloids of the effective fraction,the content of 1-deoxynojirimycin is the highest and the activity isalso the highest. So inventors prepared a refined 1-deoxynojirimycinwhich is used as a reference substance for determination. The refined1-deoxynojirimycin is characterized by NMR, MS, IR, UV and the thermalanalysis test and its structure is confirmed. The purity of refined1-deoxynojirimycin is more than 98% and meets the quality requirement ofreference substance.

Effective fractions of mulberry twig are prepared according to the abovementioned method. The said effective fraction is dissolved in water, andanalyzed by pre-column derivatization HPLC using compound (1) asreference substance. Calculated by external standard method, the contentof the total alkaloids is 50% or more in every effective fraction andthe content of compound (1), 1-deoxynojirimycin, is 27% or more in theextract. In the effective fraction, the content of compound (1) is27%÷50%=54% by weight.

Characterization of the Active Components in the Effective Fraction

The structures of the active components in the effective fraction arecharacterized by pre-column derivatization LC-MS/MS, and the resultsindicate that they are a class of polyhydroxy alkaloids, whose originalstructures are as follows:

The Satability of the Effective Fraction

The effective fractions, obtained from pilot experiments, are storedsealed at room temperature, analyzed by HPLC at different time, and theresults indicate that the quality is stable after 24 months. The resultsare listed in table 1.

TABLE 1 stability of the effective fraction obtained from pilotexperiments Content (%) No 0

3

6

12

24

1 55.0% 56.1% 54.7% 54.0% 54.5% 2 53.1% 52.6% 53.8% 54.3% 52.8% 3 57.2%55.4% 55.1% 58.3% 56.4%

The effective fraction containing alkaloids can be mixed withpharmaceutically permitted excipients to form a variety ofpharmaceutical compositions, mainly including oral dosage such astablets, dispersible tablets, chewable tablets, capsules, granules,dripping pills or oral fluid. The content of the effective fraction ofthe alkaloids in the pharmaceutical composition is 12.5-300 mg pertablet, preferably 25-100 mg per tablet, even more preferably 50-100 mgper tablet, in terms of the total alkaloids.

In order to administer drugs and improve the therapeutic effect, thedrug or the pharmaceutical composition can be administered by any knownways.

The dosage of the pharmaceutical composition can have a wide range ofchanges according to the nature and severity of the disease, thepatient's individual circumstance, the administration route, the dosageform, etc. It can be administered in a dosage unit or divided intoseveral dosage units, depending on the doctor's clinical experience, aswell as the dosage regimen, including the use of other treatments.

The pharmaceutical composition can be taken alone or with othertherapeutic drugs or symptomatic drugs. When there are synergies betweenthe compound of the invention and other therapeutic drugs, its dosageshould be adjusted to the actual situation.

In in vitro experiments, α-glucosidase inhibition percentages of theeffective fraction of this invention were determined at differentconcentrations, using glucobay(Acarbose) as positive control, and theIC₅₀ values were computed. The results showed that in vitro the totalalkaloids of mulberry twig had significant inhibitions of sucrase andmaltase, and the inhibition of sucrase was stronger than glucobay, theinhibition of maltase was equal to glucobay, while the inhibition ofamylase was significantly lower than glucobay, suggesting that thehypoglycemic activity of the effective fraction was equal to glucobay,but the side effects of gastrointestinal tract up gas might be less thanglucobay.

Using glucobay as the control, the impacts of the effective fractionfrom mulberry twig on the curves of blood glucose of normal mice afterloading sucrose and alloxan induced diabetic mice were studied. Theresults showed that at the dosage of 5-20 mg/kg in terms of the totalalkaloids, the effective fraction from mulberry twig could significantlyreduce elevated blood glucose of sucrose loaded mice, lower and postponethe peak of blood glucose, and blood glucose area under the curve wasalso significantly less than control group. This indicated that thealkaloids of the present invention could be used in preparinghypoglycemic drugs, which are used to control postprandial blood glucoseof diabetic patients.

The advantages of the present invention are as follows:

The raw material is rich, inexpensive, and its quality is controllable.

The process is simple, and the cost is low.

The hypoglycemic activity of the effective fraction of the totalalkaloids from mulberry twig was equal to glucobay, but the side effectsof gastrointestinal tract up gas might be less than glucobay.

The α-glucosidase inhibitor activity IC₅₀ of the crude extracts frommulberry twig is about 60 μg/ml, while the IC₅₀ of the effectivefraction of the total alkaloids from mulberry twig is less than 0.1μg/ml, which is much better than the crude preparations from ethanoland/or aqueous extract of mulberry twig.

DESCRIPTION OF FIGURES

FIG. 1 is a graph showing that the impact of the effective fraction frommulberry twigs on the curves of blood glucose of normal mice afterloading sucrose.

FIG. 2 is a graph showing that the impact of the effective fraction frommulberry twigs on the curves of blood glucose of alloxan induceddiabetic mice after loading sucrose.

EXAMPLES

The invention disclosed herein is exemplified by the following examplesof preparation, which should not be construed to limit the scope of theinvention.

Preparations of the Effective Fraction of the Total Alkaloids Example 1The Preparation and Content Determination of the Effective Fraction ofthe Total Alkaloids

Fresh mulberry twig was smashed and dried. To 350 kg of the driedmulberry twig, 2000 L of deionized water was added, and the mixture wasrefluxed for 2 hours. The extraction process was repeated 2 times, theextractions were combined and concentrated to 250 L, and then 700 Lethanol was added to the concentrate. The mixture was precipitated for24 hours and filtered. The supernatant fluid was added into 001×7(732#)cation exchange resin, whose dosage is crude drug: resin=1:0.2 byweight, washed with distilled water to neutral, then washed with 0.5Nammonia solution, and the eluent was collected. The eluent was addedinto 201×7(717, OH⁻) Anion exchange resin, whose dosage is crude drug:resin=1:0.6 by weight and the part not absorbed was collected andconcentrated via vacuum concentration and dried to get a light brownpowder 530 g, yield 1.51‰.

Example 2 The Preparation and Content Determination of the EffectiveFraction of the Total Alkaloids

To another batch of dried fresh mulberry twig (350 kg), 2000 L ofdeionized water was added, and the mixture was refluxed for 2 hours. Theextraction process was repeated 2 times, the extractions were combinedand concentrated to 250 L, and then 700 L ethanol was added to theconcentrate. The mixture was precipitated for 24 hours and filtered. Thesupernatant fluid was added into 001×7(732#) cation exchange resin,whose dosage is crude drug: resin=1:0.2 by weight, washed with distilledwater to neutral, then washed with 0.5N ammonia solution, and the eluentwas collected. The eluent was added into 201×7(717, OH⁻) Anion exchangeresin, whose dosage is crude drug resin=1:0.6 by weight and the part notabsorbed was collected and concentrated via vacuum concentration anddried to get a light brown powder 500 g.

Example 3 The Preparation and Content Determination of the EffectiveFraction of the Total Alkaloids

Fresh mulberry twig was smashed and dried. To 350 kg of the driedmulberry twig, 2000 L of deionized water was added, and the mixture wasrefluxed for 2 hours. The extraction process was repeated 2 times, theextractions were combined and concentrated to 250 L, and then 250 Lethanol was added to the concentrate. The mixture was precipitated for24 hours and filtered. The supernatant fluid was added into 001×7(732#)cation exchange resin, whose dosage is crude drug: resin=1:0.2 byweight, washed with distilled water to neutral, then washed with 0.5Nammonia solution, and the eluent was collected. The eluent was addedinto 201×7(717, OH⁻) Anion exchange resin, whose dosage is crude drug:resin=1:0.5 by weight and the part not absorbed was collected andconcentrated via vacuum concentration and dried to get a light brownporous powder 495 g.

Example 4 The Preparation and Content Determination of the EffectiveFraction of the Total Alkaloids

Fresh mulberry twig was smashed and dried. To 350 kg of the driedmulberry twig, 2000 L of deionized water was added, and the mixture wasrefluxed for 2 hours. The extraction process was repeated 2 times, theextractions were combined and concentrated to 250 L, and then 500 Lethanol was added to the concentrate. The mixture was precipitated for24 hours and filtered. The supernatant fluid was added into 001×7(732#)cation exchange resin, whose dosage is crude drug: resin=1:0.3 byweight, washed with distilled water to neutral, then washed with 0.5Nammonia solution, and the eluent was collected. The eluent was addedinto Dowex 1×2(OH⁻) Anion exchange resin, whose dosage is crude drug:resin=1:0.5 by weight and the part not absorbed was collected andconcentrated via vacuum concentration and dried to get a brown powder520 g.

Preparation of Dosage Form Example 1 Preparation of Tablets ofHypoglycemic Effective Fraction from Mulberry Twig

10 g of the effective fraction prepared in example 1, 5 g of calciumphosphate, 15 g of lactose, and 0.3 g of magnesium were respectivelypassed through 80 mesh sieves and mixed together. To the mixture, 70%ethanol was added to prepare damp mass, which was then granulated bypassing through 20 mesh sieves, dried, then regulated by passing through14 mesh sieves and pressed to tablets. Each tablet weighs about 300 mgand contains 100 mg of effective fraction extract, which is 50 mg oftotal alkaloids.

Example 2 Preparation of Capsules of Hypoglycemic Effective Fractionfrom Mulberry Twig

10 g of the effective fraction prepared in example 1, 5 g of calciumphosphate, 15 g of lactose, and 0.3 g of magnesium were respectivelypassed through 80 mesh sieves and mixed together. To the mixture, 70%ethanol was added to prepare damp mass, which was then granulated bypassing through 20 mesh sieves, dried, regulated by passing through 40mesh sieves and added into No. 2 hard capsules. The content of eachcapsule is about 100 mg and each capsule contains 100 mg of effectivefraction extract, which is 50 mg of total alkaloids.

Example 3 Preparation of Oral Liquid of Hypoglycemic Effective Fractionfrom Mulberry Twig

10 g of the effective fraction prepared in example 1 was dissolved anddiluted to 500 ml by distilled water, and then 0.05% of benzoic acid wasadded, flavoring agents could be selectively added, and the mixture wassub-packed in 5 ml or 10 ml of the vials and fitted with lids. Eachbottle contains 100-200 mg of effective fraction extract, which is50-100 mg of total alkaloids.

Example 4 Preparation of Dripping Pills of Hypoglycemic EffectiveFraction from Mulberry Twig

Taking 50 g of polyethylene glycol(PEG6000) and heated to melting (100°C.), and then 10 g of the effective fraction prepared in example 1 wasadded and stirred to get the molten liquid. The molten liquid was pouredto the liquid storage tank of the pill machine, and then it was addeddropwise to the liquid paraffin, cooling shaped and the paraffin waswashed away, dried. Each dripping pill weighs about 30 mg and contains 5mg of effective fraction extract, which is 2.5 mg of total alkaloids.

Example 5 Preparation Dispersible Tablets of Hypoglycemic EffectiveFraction from Mulberry Twig

10 g of the effective fraction prepared in example 1, 20 g of calciumphosphate, 5 g of lactose, 15 g of mannitol, 10 g of cross-linkedpolyethylene pyrrolidone and 0.3 g of magnesium were respectively passedthrough 80 mesh sieves and mixed together. To the mixture, 70% ethanolwas added to prepare damp mass, which was granulated by passing through20 mesh sieves, dried, then regulated by passing through 14 mesh sievesand pressed to tablets. Each tablet weighs about 600 mg and contains 100mg of effective fraction extract, which is 50 mg of total alkaloids.

Example 6 Preparation Chewable Tablets of Hypoglycemic EffectiveFraction from Mulberry Twig

10 g of the effective fraction prepared in example 1, 5 g of calciumphosphate, 5 g of lactose, 15 g of mannitol and 0.3 g of magnesium wererespectively passed through 80 mesh sieves and mixed together. To themixture, 70% ethanol was added to prepare damp mass, which wasgranulated by passing through 20 mesh sieves, dried, then regulated bypassing through 14 mesh sieves and pressed to tablets. Each tabletweighs about 300 mg and contains 100 mg of effective fraction extract,which is 50 mg of total alkaloids.

Parmalogical Experiment Example 1 In Vitro Experiment

Using glucobay(Acarbose) as positive control, α-glucosidase inhibitionpercentages of the effective fraction in example 1 were determined atdifferent concentrations, and the IC₅₀ values were computed. The resultsshowed that, in vitro, the total alkaloids of mulberry twig hadsignificant inhibitions of sucrase and maltase, and the inhibition ofsucrase was stronger than glucobay, the inhibition of maltase was equalto glucobay, while the inhibition of amylase was significantly lowerthan glucobay, suggesting that the hypoglycemic activity of theeffective fraction was equal to glucobay, but the side effects ofgastrointestinal tract up gas might be less than glucobay. The resultsare listed in table 2.

TABLE 2 α -glucosidase inhibition activity of the effective fractionIC₅₀ (ng/ml) SZ-A Arcarbose sucrase 21.9 87.1 maltase 40.4 39.3amylase >10 mcg/ml 2.4 mcg/ml

Example 2 The Impact of the Effective Fraction on the Curves of BloodGlucose of Normal Mice After Loading Sucrose

The test groups of the effective fraction set up three dosages, namely10 mg/kg, 20 mg/kg, and 40 mg/kg (p.o.), as well as a negative controlgroup (empty) and a positive control group (using glucobay as positivecontrol drug, 10 mg/kg; p.o.). The results showed that the effectivefraction of mulberry twig in the dosage of 10 mg/kg˜40 mg/1 g cansignificantly reduce elevated blood glucose of sucrose loaded mice andthe blood glucose area under the curve was also significantly less thancontrol group. The results of test groups and positive control grouphave no significant differences (see FIG. 1).

Example 3 The Impact of the Effective Fraction on the Curves of BloodGlucose of Alloxan Induced Diabetic Mice after Loading Sucrose

5 groups of alloxan induced diabetic mice were divided to negativecontrol group, positive control group using glucobay (10 mg/kg) and thevaried dosage group (10 mg/kg, 20 mg/kg, 40 mg/kg). The results showedthat the effective fraction of mulberry twig could lower and postponethe peak of blood glucose, and reduce the blood glucose area under thecurve. The effects of 20 mg/kg and 40 mg/kg treatment groups were betterthan positive control group (see FIG. 2).

1. An effective fraction of alkaloids, which is characterized by: theeffective fraction is prepared from mulberry twig and the activeingredients are a composition of alkaloids; the percentage of the totalalkaloids are 50% or more by weight in the effective fraction and thepercentage of the compound 1-deoxynojirimycin is 30% or more by weightin the total alkaloids.
 2. An effective fraction of alkaloids accordingto claim 1, which is characterized by: the effective fraction alsocontains at least one compound selected from the group consisting ofN-methyl-1-deoxynojirimycin, fagomine, 3-epi-fagomine,1,4-dideoxy-1,4-imino-D-arabinitol, 1,4-dideoxy-1,4-imino-D-ribitol,calysteginB₂, 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin,6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin and1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.
 3. Aneffective fraction of alkaloids according to any of claim 1-2, which ischaracterized by: the percentage of the compound 1-deoxynojirimycin is40% or more by weight in the total alkaloids.
 4. An effective fractionof alkaloids according to any of claim 1-3, which is characterized by:the percentage of the compound 1-deoxynojirimycin is 50% or more byweight in the total alkaloids.
 5. A method of preparing the effectivefraction of alkaloids according to claim 1-4, which is characterized byincluding the following steps: (a) mulberry twig was extracted bysolvents and the extract solution was concentrated, precipitated andremoved impurities; (b) the supernatant fluid was added into cationexchange resin and eluted with weak base eluent; and (c) the eluent wasconcentrated and added into anion exchange resin and the part notabsorbed was collected, and dried to get the effective fraction powder.6. A method according to claim 5, which is characterized by: the saidcation exchange resins are selected from sulfonic strong acid type andcarboxylic week acid type; the said anion exchange resins are selectedfrom strong base type anion exchange resin and weak base type anionexchange resins.
 7. A method according to claim 6, which ischaracterized by: the said strong base type anion exchange resin isquaternary amine (also known as quaternary amine) —NR₃OH(R ishydrocarbon group) type; the said anion exchange resins include primaryamine-NH₂ type, secondary amine-NHR type, and tertiary amine —NR₂ type.8. A method according to any of claim 6-7, which is characterized by:the materials for the said ion exchange resins include styrene series,acrylic acid series, phenolic(FP) series, epoxy(EPA) series, vinylpyridine(VP) series and urea-formaldehyde(UA) series.
 9. A methodaccording to claim 8, which is characterized by: the said styrene seriesinclude gel type and macroporous type.
 10. A method according to claim5, which is characterized by: the said weak base eluent is 0.2-1Nammonia solution; the said cation exchange resin is selected from001×7(732#) cation resin; the said anion exchange resin is Dowex1×2(OH⁻) Anion exchange resin or 201×7(717, OH⁻) Anion exchange resin.11. A method according to claim 5, which is characterized by includingthe following steps: (a) to every 350 kg of fresh mulberry twigs, 2000 Lof water is added and the mixture is refluxed for 2 hours, theextraction process is repeated 2 times, the extractions are combined andconcentrated to a volume of 250 L. 250-270 L of ethanol is added to theconcentrate. The mixture is precipitated for 24 hours and filtered; (b)the supernatant fluid is added into 001×7(732#) cation exchange resin,whose dosage is crude drug: resin=1:0.2-0.3 by weight, washed withdistilled water to neutral, then washed with 0.5N ammonia solution, andthe eluent is collected; (c) the eluent is added into Dowex 1×2(OH⁻)Anion exchange resin or 201×7(717, OH⁻) Anion exchange resin, whosedosage is crude drug: resin=1:0.5-0.6 by weight and the part notabsorbed is collected and concentrated via vacuum concentration anddried to get a light brown powder.
 12. A pharmaceutical composition withhypoglycemic activity, which is characterized by: the pharmaceuticalcomposition contains an effective dose of the effective fraction ofalkaloids according to claim 1-4 and pharmaceutically permittedexcipients.
 13. A pharmaceutical composition according to claim 12,which is characterized by: the said pharmaceutical composition includestablets, dispersible tablets, chewable tablets, capsules, granules,dripping pills or oral fluid.
 14. A pharmaceutical composition accordingto claim 13, which is characterized by: the content of the effectivefraction of alkaloids is 12.5-300 mg/tablet in terms of the totalalkaloids.
 15. A pharmaceutical composition according to claim 14, whichis characterized by: the content of the effective fraction of alkaloidsis 25-100 mg/tablet in terms of the total alkaloids.
 16. Apharmaceutical composition according to claim 15, which is characterizedby: the content of the effective fraction of alkaloids is 50-100mg/tablet in terms of the total alkaloids.
 17. The use of the effectivefraction of alkaloids according to claim 1-4 in preparing hypoglycemicagents.